In recent years, the number of patients who have been afflicted with and died of breast cancer is increasing at a significant level in Japan. Non-surgical therapeutic techniques for treating breast cancer were likely to be determined depending on estrogen receptor (ER) and progesterone receptor (PgR) expression conditions in the past. Drug therapy, such as hormonal therapy or anticancer drug therapy, is useful, and the response rate and viability have been improved. However, approximately 50% of human epidermal growth factor receptor-related 2 (HER2)-positive breast cancer cases have been hormone receptor-negative, and such cases have not been the targets of hormonal therapy. In recent years, molecular-targeted techniques, such as a trastuzumab-based antibody therapy against HER2-positive breast cancer, have been employed, the usefulness thereof has been demonstrated, and such techniques are expected as leading therapeutic methods in the future.
Breast cancer cells express a variety of growth factors and receptors thereof and form signal transduction systems associated with growth. An example thereof is the HER2 cancer gene that was identified in 1984, which is a member of the human epidermal growth factor receptor (EGFR, HER) family. A receptor is transmembrane glycoprotein which is classified as HER1, HER2, HER3, or HER4 based on structural similarities (Non-patent document 1). It is considered that the EGF receptor family contributes to signal transduction via dimer formation, and a receptor to which a ligand has bound forms a dimer with another receptor, thereby contributing to signal transduction.
HER2/neu, which defines the HER2 receptor, is considered to be a proto-oncogene, and it is present in chromosome 17 (17q21.1) (Non-patent document 2). Amplification and overexpression of the HER2/neu gene are observed in a variety of cancers, such as ovarian cancer, lung cancer, and gastric cancer, as well as in breast cancer. HER2-positive breast cancer accounts for 20% to 30% of all breast cancer cases. The expression level of this gene in adult normal tissue is very low. The natural course is specific, and recurrence takes place at an early stage when the patient would not undergo systemic treatment. Since the correlation of HER2 amplification with a poor prognosis of breast cancer was reported in 1987, a poor clinical course for HER2-positive metastatic breast cancer has been demonstrated by much research.
In 1986, it was reported that the anti-HER2 monoclonal antibody would inhibit malignant traits of neu transformed cells, which led to the development of trastuzumab targeting HER2. An mu4D5 mouse monoclonal antibody reacting with an extracellular domain of the HER2 receptor, which is a prototype of trastuzumab, exhibits activity of inhibiting growth of HER2-positive tumor cells in vitro (Non-patent document 3). Also, the antibody-dependent cell cytotoxicity (ADCC) of mouse splenic cells against SKBR3, which is a HER2-positive human breast cancer cell line, is reinforced by the mouse monoclonal antibody, and it is reported that ADCC activity also contributes to anti-tumor effects in vivo (Non-patent document 4). If a mouse antibody is subjected to clinical applications without modification, the problem of appearance of the human anti-mouse antibody (HAMA) arises. Thus, trastuzumab (trade name: Herceptin®) was prepared as a humanized antibody by transplanting only the variable region of an antigen-binding site of the mouse monoclonal antibody reacting with the extracellular domain of the HER2 receptor into the constant region of human IgG1.
HER2 excites a variety of signal transduction pathway networks including PI3K and MAPK. It is considered that trastuzumab binds to the HER2 receptor to inhibit such signal transduction pathway and it induces termination of a cell cycle, apoptosis, inhibition of angiogenesis, and the like to directly inhibit tumor cell growth. Also, inhibition of the Src tyrosine kinase, activation of PTEN involved therewith, and dephosphorylation of Akt have been reported. Further, it has been demonstrated that the Fc receptor that is expressed in immunocytes, including the NK cells, binds to the Fc region of trastuzumab, which has bound to the tumor cells, to exhibit the effects of killing tumor cells (ADCC activity) (Non-patent documents 5 to 8). Such effects of direct inhibition of tumor cell growth and ADCC activity are considered to be the main mechanisms of trastuzumab.
Trastuzumab targets HER2, and the therapeutic effects thereof significantly vary depending on HER2 protein expression level. It is known that the positive ratio significantly varies depending on assay technique and materials used. In order to inspect HER2 protein overexpression and DNA amplification, the immunohistochemical (IHC) method and fluorescence in situ hybridization (FISH) are extensively employed. Also, trastuzumab has been found to be very effective for postoperative adjuvant therapy involving a plurality of large-scale clinical testings, including the HERA test.
Based on a variety of studies that have been heretofore conducted, HER2 inhibition via trastuzumab administration was found to significantly influence the natural course of breast cancer. However, it has also been found that trastuzumab would not alter the entire natural course of HER2-overexpressing breast cancer, and it is said that the number of cases in which patients react with the first administration of trastuzumab alone is approximately one-third or lower than that for HER2-overexpressing breast cancer. In the case of microscopic metastatic cancer, similarly, it is suggested that a considerable percentage of tumors are tolerant to trastuzumab. However, the mechanism of trastuzumab tolerance has not been clearly elucidated. At present, the following possibilities are suggested as the mechanisms of trastuzumab tolerance: (1) insufficient inhibition of the HER2 extracellular region due to insufficient accession of trastuzumab; (2) a lowered HER2 expression level; (3) an altered HER2 regulator located downstream (e.g., lowered p27kip1 and quenching or inactivation of PTEN); (4) the occurrence of signal transmission by an alternate pathway (overexpression of the insulin-like growth factor I receptor (IGF1R)); and (5) lowered immunity (lowered ADCC activity, in particular).
The tolerance mechanism related to immunity of (5) above has not been actively examined. In recent years, the functions of the NK cell, which is a factor associated with ADCC activity as a main action mechanism of trastuzumab, have become elucidated. Inhibitory receptors are expressed in NK cells, and the killer cell lectin-like receptor G1 (KLRG1) was identified as one such inhibitory receptor. Most of the ligands of inhibitory receptors that have been found with regard to NK cells were associated with the MHC class I molecule. In 2006, M. Ito et al. reported that the KLRG1 ligand was not of the MHC class I molecule.
KLRG1 was discovered as a functional molecule expressed in the RBL-2H3 rat basophilic leukemia cell line (MAFA: the mast cell function-associated antigen) (Non-patent document 9). KLRG1 crosslinks with the anti-KLRG1 antibody in the RBL-2H3 cell, and the degranulation reaction caused by Fc receptor stimulation is inhibited. As a result of cDNA cloning of rat KLRG1, it has been reported by Pecht et al. that KLRG1 is a homodimer of the type II transmembrane protein having a C-type lectin-like structure in the extracellular region and an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the intracellular region (Non-patent document 10). KLRG1 is expressed in some NK or T cells in the case of humans and mice (Non-patent documents 11 to 13). In contrast, it is known that expression thereof is observed in approximately 50% of the peripheral blood NK cells of healthy individuals.